Membrane based electrochemical test device and related methods

ABSTRACT

An electrochemical test device is provided for determining the presence or concentration of an analyte in an aqueous fluid sample. The electrochemical test device includes a working electrode and a counter electrode made of an amorphous semiconductor material. The working electrode is overlaid with a reagent capable of reacting with an analyte to produce a measurable change in potential which can be correlated to the concentration of the analyte in the fluid sample. The test device optionally contains a reference electrode made of an amorphous semiconductor material having a reference material on the reference electrode.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to an electrochemical test devicesuitable for determining the presence or concentration of chemical andbiochemical components (analytes) in aqueous fluid samples and bodyfluids such as whole blood or interstitial fluid. Additionally, thisinvention relates to a method of using such test devices for determiningthe presence or concentration of an analyte and to processes forpreparing such a test devices.

[0003] 2. State of the Art

[0004] Medical studies have demonstrated that the incidence of seriouscomplications resulting from diabetes, such as vision loss and kidneymalfunction, can be significantly reduced by careful control of bloodglucose levels. As a result, millions of diabetics use glucose testingdevices daily to monitor their blood glucose concentrations.Additionally, a wide variety of other blood testing devices are used todetermine the presence or concentration of other analytes, such asalcohol or cholesterol, in aqueous samples, such as blood.

[0005] Such blood testing devices typically employ either a drychemistry reagent system or an electrochemical method to test for theanalyte in the fluid sample. In recent years, electrochemical testingsystems have become increasingly popular due to their small size andease of use. Such electrochemical testing systems typically useelectrochemistry to create an electrical signal which correlates to theconcentration of the analyte in the aqueous sample.

[0006] Numerous electrochemical testing systems and related methods areknown in the art. For example, European Patent Publication No. 0 255 291B1, to Birch et al., describes methods and an apparatus for makingelectrochemical measurements, in particular but not exclusively for thepurpose of carrying out microchemical testing on small liquid samples ofbiological, e.g. of clinical, origin.

[0007] European Patent Publication No. 0 351 891 B1, to Hill et al.,teaches a method of making an electrochemical sensor by printing. Thesensor is used to detect, measure or monitor a given dissolved substratein a mixture of dissolved substrates, most specifically glucose in bodyfluid.

[0008] U.S. Pat. No. 5,391,250, to Cheney II et al., teaches a method offabricating thin film electrochemical sensors for use in measuringsubcutaneous or transdermal glucose. Fabrication of the sensorscomprises placing a thin film base layer of insulating material onto arigid substrate. Conductor elements for the sensors are formed on thebase layer using contact mask photolithography and a thin film coverlayer.

[0009] U.S. Pat. No. 5,437,999, to Diebold et al., teaches a method offabricating thin film electrochemical devices which are suitable forbiological applications using photolithography to define the electrodeareas. The disclosures of each of the above patent specifications areincorporated herein by reference in their entirety.

[0010] An excellent reference on materials and process for fabricatingelectronic components is Charles A. Harper, Handbook of Materials andProcesses for Electronics, 1984, Library of Congress card number76-95803. It provides detail process information on thick film, thinfilm and photo resist processes.

[0011] Existing electrochemical testing systems, however, have certainlimitations from the perspective of the end user or the manufacturer.For example, some electrochemical testing systems are difficult orcostly to manufacture. As a result, such devices are too expensive to beused on a daily basis by, for example, diabetics. Other electrochemicaltesting systems are not sufficiently accurate to detect certain analytesat very low concentrations or to give reliable measurements of theanalyte's concentration. Additionally, many electrochemical devices aretoo large to be easily carried by those needing to test their blood on aregular basis throughout the day. Thus, a need exists for improvedelectrochemical test devices.

SUMMARY OF THE INVENTION

[0012] The present invention utilizes amorphous semiconductor appliedwith thin film manufacturing techniques and membranes which have a skinon each planar surface to provide an electrochemical test devicesuitable for determining the presence or concentration of analytes inaqueous fluid samples. By using amorphous semiconductor materialsapplied with film manufacturing techniques and dual skin membranes,uniform electrochemical test devices having well-defined reproducibleelectrode areas can be manufactured economically.

[0013] In particular, the test devices of this invention have veryuniform surface areas which reduce the variability of theelectrochemical test. In this regard, it has been found that the surfacearea of the electrodes and the accuracy of applying the reagent arecritical to producing an accurate test. If the surface area is notconsistent from test to test then each of the test devices must beindividually calibrated to insure accurate readings. The test devices ofthe present invention permit highly accurate electrochemical analytemeasurements to be performed on very small aqueous fluid samples withoutthe need for individual calibration of each test device. The presentinventions provide for the accurate reproduction of the test devices byusing controlled deposition methods, such as sputtering or vapordeposition and smooth skin membranes to form the electrodes withconsistent size and surface morphology from device to device incontinuous production. These devices can also be readily manufactureddue to the lower cost and the flexible nature of the amorphoussemiconductor materials which facilitates production by continuous rollprocessing versus the step and repeat printing methods currentlyemployed. The ability to use continuous processing to fabricate thedevice, such as continuous processes utilizing continuous roll coating,continuous roll sputtering, continuous systems utilizing contact masks,results in high volume manufacturing capability and substantial costreductions over the step and repeat processes. Additionally, theamorphous nature of the conductors electrodes and constructed and usedaccording to this invention eliminates problems found in prior testdevices which utilize conventional conductor and semiconductormaterials, which are crystalline in nature or are noble metals and, as aresult, require flat and rigid substrates to prevent cracking duringmanufacture, distribution or use. The membrane in the present inventionis the earlier for the indicating reagent and forms the surface for theelectrode formation. The membrane comprises the matrix in which thereagent is carried or impregnated and comprises the two exterior skinsurfaces on which the electrodes are placed. The skin surfaces can besmooth skin suitable for carrying the electrodes and can be porous topass the aqueous fluid samples or can have pores sized to screen orblock selected components from aqueous fluid sample, such as red bloodcells in a blood sample.

[0014] Dry electrochemical test devices fall into two primaryconfigurations. The first configuration utilizes two electrodes, i.e., aworking electrode and a counter electrode. The second configurationutilizes three electrodes, i.e., a working electrode, a counterelectrode and a reference electrode. The use of the reference electrodeand a reference material provides a fixed reference for the test. Thetest devices of the present invention are well suited for a twoelectrode system however, a contact mask could be employed duringsputtering to create a surface with two electrodes.

[0015] Accordingly, in one of its aspects, the present inventionprovides an electrochemical test device for determining the presence orconcentration of an analyte in an aqueous fluid sample, saidelectrochemical test device comprising:

[0016] (a) a nonconductive surface;

[0017] (b) a working electrode comprising an amorphous semiconductormaterial affixed to the non-conductive surface of a double skinmembrane, said working electrode having an first electrode area, a firstlead and a first contact pad;

[0018] (c) a counter electrode comprising an amorphous semiconductormaterial affixed to the opposite nonconductive surface of a double skinmembrane, said counter electrode having an second electrode area, asecond lead and a second contact pad; and

[0019] (d) a reagent capable of reacting with the analyte to produce ameasurable change in potential which can be correlated to theconcentration of the analyte in the fluid sample, said reagent beingimbibed into the membrane matrix between the two electrode surfaces.

[0020] In another embodiment of this invention, the test device furthercomprises a reference electrode comprising an amorphous semiconductormaterial affixed to the non-conductive surface, said reference electrodehaving an electrode area, a lead, and a contact pad, and wherein atleast a portion of the electrode area is overlaid with a referencematerial. Preferably, the reference material is a silver/silver chloridelayer, a mercury/mercury chloride layer or a platinum/hydrogen material.This electrode could be either the counter electrode or an independentthird electrode.

[0021] In a preferred embodiment of this invention, the test devicefurther comprises a blood separating membrane with two skin surfaces.The blood separating membrane separates whole blood samples into highlycolored and relatively clear fluid portions before analysis. The bloodseparating membrane effectively blocks or filters red blood cells andallows essentially clear fluid to pass to the test reagent imbibed inthe membrane matrix. As a result, the analyte is measured in the clearfluid portion of the sample contacting the electrodes therebysubstantially eliminating the red blood cells from the reaction andminimizing interference from the cells present in blood. This embodimenthas the additional benefit of keeping the test site from drying out andthereby improves the performance of test devices designed for smallsample sizes, such as in the 1 to 5 microliter range.

[0022] Preferably, the membrane is selected from polysulphone,polyethersulphone, or nylon and is cased with a tight pore skin on eachside and a relatively isotropic matrix between each skin surface

[0023] The skin pore size is relatively tight approximately 0.1 to 0.5microns in size and the isotropic matrix being 0.5 microns or greater inpore size. The tight pore size provides a uniform surface morphology onwhich the amorphous semiconductor electrodes are formed according tothis invention. Better surface morphology of the membrane results in amore consistent surface for the amorphous semiconductor electrodes. Thisprovides improved accuracy of test results and consistency ofperformance.

[0024] Preferably, the amorphous semiconductor material used in thisinvention is amorphous silicon oxide. More preferably, the amorphoussilicon oxide is doped with lithium, potassium, or a similar conductingion to increased conductivity. Doping with lithium is particularlypreferred. However, amorphous carbon, gold, silver or other conductormaterials which do not interfere with the electrochemistry of thereagent system are also suitable. The amorphous semiconductor materialcan be applied by using various techniques including sputtering,evaporation, vapor phase deposition or other thin film depositiontechnique to form a conductive layer on the membrane surface The surfacetexture of the amorphous semiconductor material is preferably less than13 micro inches or 0.33 microns. However, rougher textures can be useddepending on the accuracy of the desired test device.

[0025] The reagent employed in the electrochemical test device istypically selected based on the analyte to be tested and the desireddetection limits. The reagent preferably comprises an enzyme and a redoxmediator. When the analyte to be detected or measured is glucose, theenzyme is preferably glucose oxidase and the redox mediator is potassiumferrocyanide.

[0026] The electrochemical test device of the present invention is usedto determine the presence or concentration of an analyte in an aqueousfluid sample. Accordingly, in one of its method aspects, the presentinvention provides a method for determining the presence orconcentration of an analyte in an aqueous fluid sample, said methodcomprising:

[0027] (a) providing an electrochemical test device comprising:(i)double skinned membrane; (ii) a working electrode comprising anamorphous semiconductor material affixed to the membrane surface, saidworking electrode having an first electrode area, a first lead and afirst contact pad area; (iii) a counter electrode comprising anamorphous semiconductor material affixed to the non-conductive surface,said counter electrode having a second electrode area, a second lead,and a second contact pad; and (iv) a reagent capable of reacting withthe analyte to produce a measurable change in potential which can becorrelated to the concentration of the analyte in the fluid sample, saidreagent being imbibed into the membrane matrix between the two electrodesurfaces;

[0028] (b) inserting the electrochemical test device into a meterdevice;

[0029] (c) applying a sample of an aqueous fluid to the membrane area ofthe working electrode;

[0030] (d) reading the meter device to determine the presence orconcentration of the analyte in the fluid sample.

[0031] In another embodiment, the test device employed in this methodfurther comprises a reference electrode comprising an amorphoussemiconductor material affixed to the counter electrode membranesurface, said reference electrode having a third electrode area, a thirdlead, and a third contact pad, and wherein at least a portion of thethird electrode area is overlaid with a reference material.

[0032] Preferably, the reference material is a silver/silver chloridelayer, a mercury/mercury chloride layer or a platinum/hydrogen material.Silver/silver chloride is a particularly preferred reference material.The separation of the counter electrode conductive path and referenceelectrode is accomplished by using a mask to create the differentgeometries on the same surface.

[0033] Preferably, the membrane is selected from polysulphone,polyethersulphone, or nylon and is cased with a tight pore skin on eachside and a relatively isotropic matrix between each skin surface

[0034] The skin pore size is relatively tight approximately 0.1 to 0.5microns in size and the isotropic matrix being 0.5 microns or greater inpore size. The tight pore size provides a uniform surface morphology onwhich the amorphous semiconductor electrodes are formed according tothis invention. Better surface morphology of the membrane results in amore consistent surface for the amorphous semiconductor electrodes. Thisprovides improved accuracy of test results and consistency ofperformance.

[0035] As discussed above, the present invention utilizes amorphoussemiconductor materials and thin manufacturing techniques to provideelectrochemical test devices. Thin film technologies can be used tocreate the amorphous semiconductor material conductive layers andelectrodes according to this invention. Accordingly, in one of itsprocess aspects, the present invention provides a process for preparingan electrochemical test device suitable for determining the presence orconcentration of an analyte in an aqueous fluid sample, said processcomprising the steps of:

[0036] (a) providing a skinned membrane having a first and an oppositesecond surface;

[0037] (b) depositing an amorphous semiconductor material on said firstsurface to form a conductive layer,

[0038] (c) depositing an amorphous semiconductor material on saidopposite second surface to form a conductive layer

[0039] (d) applying a reagent to the membrane which is imbibed into themembrane matrix between the two surfaces, said reagent being capable ofreacting with an analyte in an aqueous fluid sample to produce ameasurable change in potential which can be correlated to theconcentration of the analyte in the fluid sample.

[0040] In another embodiment, step (c) of this process further comprisesforming a reference electrode comprising a third electrode having athird electrode area, a third lead and a third contact pad. This isaccomplished by using a mask to form the two distinct electrodes and ina step (e) forming a silver chloride surface.

[0041] In a preferred embodiment, step (a) above comprises the steps of:

[0042] (f) providing a double skinned membrane with the correct poresize distribution; and

[0043] In another preferred embodiment, step (d) above comprises thesteps of:

[0044] (h) positioning a mask on the opposing membrane surface andsputtering an amorphous conductive surface to the membrane surface toform independent electrodes;

[0045] (i) said second exposed conductive surface area comprising (i) acounter electrode comprising a second electrode having a secondelectrode area, a second lead and a second contact pad, and optionally(ii) a reference electrode comprising a third electrode having a thirdelectrode area, a third lead and a third contact pad.

[0046] In further preferred embodiment, step (d) above further comprisesthe steps of:

[0047] (o) positioning a second mask on the opposing surface so that theopposing electrode area is masked and the third reference electrode areais exposed;

[0048] (p) applying a reference material the third electrode area;

[0049] (q) removing the mask.

[0050] Preferably, the process employed to prepare the test devices ofthis invention is a continuous process. The ability to use continuousprocessing to fabricate the test devices, such as a continuous processutilizing continuous roll coating, continuous roll sputtering,continuous sputtering systems utilizing contact masks, results in highvolume manufacturing capability and substantial cost reductions over thestep and repeat printing processes.

BRIEF DESCRIPTION OF DRAWINGS

[0051]FIG. 1 illustrates a plan view of the membrane showing the topside and amorphous semiconductor coating.

[0052]FIG. 2 illustrates a side view of the membrane showing the skinand amorphous semiconductor coatings.

[0053]FIG. 3 illustrates a plan view of the membrane showing the bottomside and amorphous semiconductor coating.

[0054]FIG. 4 illustrates an isometric view of the non conductive toplayer.

[0055]FIG. 5 illustrates an isometric view of the double skin membranewith conductive layers.

[0056]FIG. 6 illustrates an isometric view of the non conductive bottomlayer.

[0057]FIG. 7 illustrates an isometric view of the assembled device.

[0058]FIG. 8 illustrates finished reference and opposing electrode.

[0059]FIG. 9 illustrates an isometric view of membrane position in asputter frame.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention utilizes amorphous semiconductor materialsand double skin membranes to provide an electrochemical test devicesuitable for determining the presence or concentration of analytes inaqueous fluid samples. By using amorphous semiconductors and the doubleskinned membranes, uniform electrochemical test devices havingwell-defined reproducible electrode areas can be manufacturedeconomically. These areas have very uniform surface morphology whichreduces the variability of the electrochemical test. The surfacemorphology is directly related to the surface area of the electrodes.The amount and concentration of applied reagents are also critical toproducing an accurate test. If the surface area is not consistent fromdevice to device then the individual devices manufactured have to beindividually calibrated to insure accurate readings in a meter. The testdevices of the present invention permit highly accurate electrochemicalanalyte measurements to be performed on very small aqueous fluid samplesdue to the accurate reproduction of the test devices using controlleddeposition methods such as sputtering, vapor phase deposition, glowdischarge methods or evaporation and the very uniform surface of thedouble skin membranes to form the geometries. These devices can also bereadily manufactured due to the lower cost and flexible nature of theamorphous semiconductor materials which allow the use of volumeproduction in continuous processing manufacturing methods.

[0061] Thin film technologies are preferred for forming the amorphoussemiconductor material layer or coating, such as sputtering, vapor phasedeposition, glow discharge methods or evaporation. Such processes arecapable of producing coatings up to a thickness of a few microns and ofapplying the coatings uniformly over the entire surface. If desired,thick film technologies are capable of producing much thicker layers inthe range of less than 0.005 inches. While both methods can produceuniform surfaces, thin film surface morphology is dependent on thesurface roughness of the underlying substrate, whereas the thick filmtechnologies are capable of changing the final surface morphology of thedevice due to their thicker structure.

[0062] The electrochemical test device of this invention employs atleast two types of electrodes. The first type of electrode is referredto as the “working or indicating electrode” and the second type isreferred to as the “counter or opposing electrode”. These electrodespermit changes in potential between the electrodes to be accuratelymeasured when the analyte is applied across the electrodes, and thereagent reactive with the analyte is bound to the membrane matrixbetween the electrodes formed on the skin. Optionally, the test devicemay also contain a reference electrode. The reference electrode allowsaccurate measurements of potential to be made with respect to a fixedreference point. Any suitable reference electrode may be used, includesilver/silver chloride, mercury/mercury chloride and platinum/hydrogenreference electrodes. Preferably, the reference electrode is asilver/silver chloride reference electrode and the working electrode hasa potential ranging from about +1 to −1 volts versus the referenceelectrode. Reference electrodes suitable for use in this invention arefurther described in R. S. C. Cobbold, Transducers for BiomedicalMeasurements: Principles and Applications, pp. 343-349, John Wiley &Sons (New York).

[0063] The electrodes in the test device of this invention aremanufactured using a double skinned membrane. Any suitable dielectricmaterial may be used as the material for the membrane matrix as long asa tight pore skin can be formed on both sides of the membrane. Forexample, polysulphone, polyethersulphone, or nylon and is casted with atight pore skin on each side and a relatively isotropic matrix betweeneach ski surface. The skin pore size is relatively tight approximately0.1 to 0.5 microns in size and the isotropic matrix being 0.5 microns orgreater in pore size. Such polymeric materials are well known in the artand are commercially available. The membranes employed in preferredembodiments of this invention is Supor 200 available from Pall GelmanSciences

[0064] The electrodes are then prepared on the membrane usingsemiconductor manufacturing techniques. Typically, a conductive layer isfirst deposited on or applied to the membrane. The conductive layercomprises an amorphous semiconductor material, such as amorphous siliconoxide, amorphous carbon, gold or silver and the like. Amorphous metalscan be formed by permitting a small amount of helium gas to escape inthe sputtering chamber during sputtering. Such materials are well knownin the art. For example, formation of suitable amorphous semiconductorfilms are described in U.S. Pat. No. 4,226,898, to Ovshinsky, thedisclosure of which is incorporated herein by reference in its entirety.

[0065] Preferably, the amorphous semiconductor material employed in thisinvention is doped with lithium, potassium, or a similar conducting ionto increased conductivity. U.S. Pat. Nos. 3,983,076, and 4,178,415, toOvshinsky, describe suitable methods for adding various compounds toamorphous semiconductor material to increase its conductivity. Thedisclosures of these patents are incorporated herein by reference intheir entirety.

[0066] The amorphous semiconductor material is preferably deposited onthe non-conducting membrane skin to form a conductive layer having athickness of less than about 0.005 inches, more preferably a thicknessranging from about 1 to about 5 microns. The amorphous semiconductormaterial may be applied to the membrane using any art recognizedprocedure to form a conductive layer, such as sputtering, vapor phasedeposition, glow discharge deposition, evaporation and the like.Alternatively, other deposition techniques such as electroplating, thickfilm laminating, roll transfer and the like may be employed. Thinnerfilms are best achieved by sputtering, evaporation, vapor phasedeposition, glow discharge methods or other thin film deposition methodwhereas thicker films are best formed using thick film depositiontechniques such as electroplating, thick film laminating, roll transferand the like. Charles A. Harper, Handbook of Materials and Processes forElectronics, 1984, (Library of Congress card number 76-95803) discussesvarious methods of forming thin films in Chapter 11, describes variousthick film process in Chapter.

[0067] Preferably, the amorphous semiconductor material is depositedusing vapor phase deposition or sputtering techniques. In a particularlypreferred embodiment, the amorphous semiconductor material is formed byco-deposition of the amorphous semiconductor material and the dopingmaterial such as by co-vacuum deposition or co-sputtering. Co-sputteringcan be accomplished in a conventional DC or RF. sputtering system, suchas available from ULVAC Vacuum Systems. In this process, a cathode ortarget is bonded to a standard aluminum backing plate and is made of thesemiconductor material to be deposited. Also, pieces of the dopingmaterial are secured to the cathode or target. Substrates upon which theamorphous semiconductor material and the doping material are to bedeposited are carried by a holder located from the target by a distanceof about 3.5 cm for a 3 ⅓″ diameter cathode or passed continuously bythe target.

[0068] The sputtering machine is first evacuated to a vacuum pressuresomewhat less than about 6×10⁻⁶ torr to provide a background vacuumpressure. Argon is bled into the machine to provide an operatingpressure of about 5×10⁻³ torr as determined by a reading on a Piranivacuum gauge, giving an actual vacuum pressure of about 7×10⁻³ torr inthe machine. The surface of the cathode or target and pieces of dopingmaterial are first cleaned by sputtering against the shutter of themachine adjacent to substrates for about 30 minutes. Thereafter, theshutter is opened and the semiconductor material of the target and thepieces of doping material on the target are co-sputtered onto thenon-conductive coating or substrate. The cathode or target and theholder for the substrates are both water cooled so that theirtemperature is low during the sputtering operation, as for example,approximately 50° C. The RF.power of the machine may have a frequency ofabout 13.56 MegaHertz, and about 1000 Volts of forward power, about50-70 Watts being utilized for 3.5″ diameter cathode or target.

[0069] The deposition rates depend upon the materials being sputtered,and the time of deposition is varied to obtain desired thicknesses ofthe deposit. The thicknesses of the simultaneously deposited amorphoussemiconductor film having the doping material therein may vary from afew hundred anastroms to about 5 microns (5 μ), depending upon the usesto which the amorphous semiconductor film is to be put. The amorphoussemiconductor material is deposited on the membrane.

[0070] The amount of doping material homogeneously incorporated in theamorphous semiconductor material is generally determined by the area ofthe pieces of the doping material applied to the semiconductor materialof the cathode or target. Desired percentage of modifier material addedto the amorphous semiconductor material may accordingly be convenientlycontrolled. By utilizing co-sputtering as generally here described, thedoping material is substantially homogeneously incorporated in thesemiconductor material to provide the beneficial features of thisinvention.

[0071] Preferably, the amorphous semiconductor material is appliedcontinuously to the membrane using art recognized continuous processes.Such continuous processes are described, for example, in U.S. Pat. No.3,983,076, to Rockstad, and in U.S. Pat. Nos. 3,271,591 and 4,178,415,to Ovshinsky, the disclosures of which are incorporated herein byreference in their entirety. The ability to use continuous processing tofabricate the test devices of this invention, such as a continuousprocess utilizing continuous roll coating, continuous roll sputtering,results in high volume manufacturing capability and substantial costreductions over the step and repeat processes currently used. Suitablemachines for such continuous processes are made by Ulvac Vacuum Systemsof Japan.

[0072] Optionally, if a reference electrode is used, the thirdelectrode, if present, is constructed by using a contact mask whichisolates the two electrodes formed on the bottom skin. The contact maskpermits only application of sputtered material in the open areas anddoes not permit the coating on the covered areas. The referenceelectrode is then converted into a reference electrode by applying asuitable reference material. Suitable reference materials includesilver/silver chloride, a mercury/mercury chloride and platinum/hydrogenmaterials. Such materials can be applied to the third electrode area ofthe reference electrode by any thin film deposition method describedabove.

[0073] A particularly preferred reference material is silver/silverchloride. In order to obtain consistent results from device to device,the silver/silver chloride layer must be applied such that thesilver/silver chloride layer covers essentially the same surface area ineach device manufactured. Preferably, this is accomplished using contactmask as described above to expose a precise area of the electrode afterdeveloping. A thin layer of silver is then deposited on the exposedarea, preferably by sputtering or other consistent film applicationmethod. The silver layer is preferably less than about 0.001 inches inthickness, more preferably about 1 micron to about 5 microns inthickness. Alternatively, a silver layer can be applied to the electrodearea of the reference electrode using pad printing, inkjet methods,transfer roll printing or similar techniques. The silver layer is thencontacted with FeCl₃ solution to convert the silver surface to silverchloride thereby forming a silver/silver chloride layer

[0074] The electrochemical test device is then completed by applying anappropriate reagent to the membrane. The reagent is imbibed into themembrane matrix under the sample application area. Suitable reagents fordetermining the presence or concentration of various analytes are wellknown in the art and are described in further detail herein below.

[0075] Preferred Process for Preparing the Electrodes

[0076] As described above, the electrodes for the electrochemical testdevice of this invention are prepared using amorphous semiconductormaterials and double sided skinned membrane. Preferably, such electrodesare formed in a continuous manner. A preferred process for thepreparation of electrodes suitable for use in this invention isillustrated in FIGS. 1-9.

[0077] As illustrated in FIGS. 1, 2 and 3, the membrane 1 which hasskins 12 and 13 is coated with amorphous semiconductor material creatingsurfaces 2 and 4 which are the electrodes have a thickness of less thanabout 0.005 inches and preferable in the range of 1 to 5 microns.

[0078]FIG. 4 shows an isometric view of the top non conductive layer 4which has been coated with adhesive 7. Non conductive layer 4 has holes5 and 6 punched in the layer. Hole 5 is the sample application hole andpermits the application of the indicating reagent during manufacture.Hole 6 is permits the measuring device to contact the conductive surfaceof the membrane.

[0079]FIG. 5 shows an isometric view of the membrane with conductivelayers 2 and 4 and reference coating 14 (not shown) applied to theopposite side of the membrane.

[0080]FIG. 6 shows an isometric view of the bottom non conductive layer10 which has been coated with adhesive 8. Non conductive layer 10 hasholes 9 and 11 punched in the layer. Hole 9 is the sample vent hole andpermits the venting of the air in the membrane during application of theindicating reagent when the strip is manufactured. Hole 11 is permitsthe measuring device to contact the conductive surface of the membrane.

[0081]FIG. 7 is an isometric view of the finished device showing themembrane 1 sandwiched between the top and bottom non conductive layers 4& 10 with top vent hole 5 and top contact hole 6.

[0082] As shown in cross section FIG. 8 is a bottom view of a membraneshowing opposing electrode 3 and reference electrode 15 with referencecoating 14. The pattern is formed by sputtering on the skin and using acontact mask to pattern the electrodes.

[0083] As illustrated in FIG. 9, the membrane 1 can be loaded into aframe 20 to permit discrete manufacturing.

[0084] Reagents

[0085] Various types of analytical or electrochemical sensor reagentsmay be applied to the electrodes. To create a functional electrochemicaltest device, a reagent chemistry must be selected based on the analyteto be tested and the desired detection limits. Preferably, the reagentis deposited on the membrane such that a uniform amount is applied fromsensor to sensor. The reagent may be applied using any conventionalprocedure, such as nozzle coating through the hole 5 in layer 4 usingIVEK pumps or any other drop on demand system capable of deliveringconsistent and uniform volume of reagent.

[0086] The reagent is applied through the working electrode, but may insome cases also be applied through the other electrodes. After thereagent has been imbibed into the membrane, it is typically dried.Subsequently, when the test device is used, the test sample of aqueousfluid, such as blood, rehydrates the reagent and a potential is appliedto the electrodes from which a current measurement may be taken by ameter. The membrane matrix acts as reagent carrier and electrode spacer.This permits the device to have a reduced manufacturing cost due to theexcellent uniformity of the membrane thickness and isotropic nature ofthe inner matrix of the membrane.

[0087] An example reagent formulation suitable for use in the presentinvention is described below. This reagent may be used to determine thepresence or concentration of glucose in an aqueous fluid sample.Preferably, this reagent formulation is used with an electrochemicalsensor having an opposing electrode 3, working electrode 2 and referenceelectrode 15. Reagent Formulation Amount/ Material Concentration2-(N-morpholino)ethanesulfonic acid (MES buffer) 100 millimolar (mM)Triton X-100 0.08% wt/wt Polyvinyl alcohol (PVA) mol. wt. 10K 88% 1.00%wt/wt hydrolyzed Imidazole osmium mediator, reduced, as defined in 6.2mM U.S. Pat. No. 5,437,999 Glucose Oxidase 6000 units/mL

[0088] The above reagent formulation may be prepared using the followingprocedures:

[0089] (a) 1.952 grams of MES buffer is added to 85 mL of nanogradewater. The mixture is stirred until the components dissolve. The pH ofthe solution is adjusted to 5.5 with NaOH. The volume of the solution isthen brought to 100 mL of final buffer solution.

[0090] (b) 0.08 grams of Triton X-100 and 1 gram of PVA is added to abeaker capable of holding all the components (e.g., a 200 mL beaker).The buffer solution is added to bring the total solution weight to 100grams. The mixture is heated to boiling and stirred to dissolve the PVA.

[0091] (c) 4.0 mg of the reduced osmium mediator is added to 1 mL of thesolution from step (b) above. The mixture is stirred to dissolve themediator.

[0092] (d) The mixture is left to cool to room temperature.

[0093] (e) 6000 units of glucose oxidase are added and the mixture ismixed until the enzyme is dissolved.

[0094] The above reagent formulation may be used to determine thepresence or concentration of glucose in an aqueous fluid sample. As willbe apparent to those skilled in the art, other reagent formulations maybe employed to assay different analytes. Such reagent formulations arewell known in the art. Typically, such reagent formulations are designedto react specifically with the desired analyte to form a measurableelectrochemical signal.

[0095] Without being limited to theory, it is believed that in theexample reagent formulation described above, glucose is anaerobicallyoxidized or reduced with the involvement of the enzyme and the redoxmediator. Such a system is sometimes referred to as an amperometricbiosensor. Amperometry refers to a current measurement at constantapplied voltage on the working electrode. In such a system, the currentflowing is limited by mass transport. Therefore, the current isproportional to the bulk glucose concentration. The analyte, enzyme andmediator participate in a reaction where the mediator is either reduced(receives at least one electron) or oxidized (donates at least oneelectron). The glucose reaction ends when glucose oxidase is oxidizedand the mediator is reduced. The mediator is then oxidized at thesurface of the working electrode by the applied potential difference.Changes in the system amperage result from changes in the ratio ofoxidized/reduced form of the redox mediator. The amperage changedirectly correlate to the detection or measurement of glucose in thetest sample.

[0096] Various enzymes may be used in the reagent formulations employedin this invention. The particular enzyme employed will vary depending onthe analyte to be detected or measured. Preferred enzymes includeglucose oxidase, glucose dehydrogenase, cholesterol esterase and alcoholoxidase. The amount of enzyme employed will generally range from about0.5 to about 3.0 million units of enzyme per liter of reagentformulation.

[0097] The reagent formulation will also typically contain a redoxmediator. The redox mediator will generally be chosen to be compatiblewith the enzyme employed and combinations of redox mediators and enzymesare well known in the art. Suitable redox mediators include, by way ofexample, potassium ferricyanide and ferrocene derivatives, such as1,1¢-dimethyl ferrocene. The amount of redox mediator employed in thereagent formulation will typically range from about 0.15M to about 0.7M.Additional mediators suitable for use in this invention includemethylene blue, p-benzoquinone, thionine, 2,6-dichloroindophenol,gallocyanine, indophenol, polyviologen, osmium bis(2,2¢-bipyridine)dihydrochloride, and riboflavin-5¢-phosphate ester. Optionally, thesemediators can be chemically bound or entrapped in a matrix, such as apolymer, using procedures well known in the art

[0098] Examples of enzyme/mediator combinations suitable for use in thisinvention include, but are not limited to, the following: Analyte EnzymeMediator glucose glucose dehydrogenase ferricyanide glucose glucoseoxidase tetracyanoquinodimethane cholesterol cholesterol esteraseferricyanide alcohol alcohol oxidase phenylenediamine

[0099] A preferred reagent chemistry uses potassium ferricyanide as amediator.

[0100] In addition to an enzyme and a redox mediator, the reagent layeron the electrode preferably further comprises a buffer, a stabilizer, adispersant, a thickener or a surfactant. These materials are typicallyemployed in amounts which optimize the reaction of the reagents with theanalyte. The concentration ranges for these components referred to beloware for the reagent formulation before it has dried on the electrodesurface.

[0101] A buffer is preferably employed in the reagent formulation toprovide a satisfactory pH for enzyme function. The buffer used must havea higher oxidation potential than the reduced form of the redoxmediator. A preferred buffer for use in this invention is a phosphatebuffer having a concentration ranging from about 0.1M to about 0.5M.Other suitable buffers include BES, BICINE, CAPS, EPPS, HEPES, MES,MOPS, PIPES, TAPS, TES and TRICINE buffers (collectively known as ‘GOOD’buffers), citrate, TRIS buffer, and the like. The ‘GOOD’ and TRISbuffers are commercially available from Sigma-Aldrich, Inc. (St Louis,Mo., U.S.A.).

[0102] A stabilizer may also be employed in the reagent formulation tostabilize the enzyme. When the enzyme used is glucose oxidase, apreferred stabilizer is potassium glutamate at a concentration rangingfrom about 0.01 to 4.0% weight. Other suitable stabilizers includesuccinate, aspartate, blue dextran and the like.

[0103] Additionally, dispersants may be used in the reagent formulationto enhance the dispersion of the redox mediator and to inhibit itsrecrystallisation. Suitable dispersants include microcrystallinecellulose, dextran, chitin and the like. Typically, the dispersant isused in the reagent formulation in an amount ranging from about 1.0 toabout 4.5% weight. Preferred dispersants include, but are not limitedto, AVICEL RC-591 (a microcrystalline cellulose available from FMCCorp.) and NATROSOL-250 M (a microcrystalline hydroxyethylcelluloseavailable from Aqualon).

[0104] A thickener may also be employed in the reagent formulation tohold the reagent to the electrode surface. Suitable thickeners includewater soluble polymers, such as polyvinylpyrrolidone.

[0105] Additionally, a surfactant may be added to the reagentformulation to facilitate rapid and total wetting of the electrodesurface. Preferably, the reagent formulation contains a nonionicsurfactant in an amount ranging from about 0.01 to 0.3% by weight. Apreferred surfactant is TRITON X-100, available from Sigma-Aldrich, Inc.

[0106] Use of the Electrochemical Test Device

[0107] To illustrate the use of an electrochemical test device of thisinvention, the following glucose assay is described. It will beunderstood, however, that by selecting the proper reagent, otheranalytes may be determined using these procedures.

[0108] The electrodes of the electrochemical test device are prepared asdescribed above and the membrane is coated with 1.0 μL of theabove-described reagent formulation and dried.

[0109] The electrochemical test device is then inserted in a meterbefore the test sequence is initiated Any suitable meter device whichhas contacts that interface with the test device contacts may beemployed. Such metering devices are well known in the art. The meterwill generally contain a measuring circuit and be adapted to apply analgorithm to the current measurement whereby the analyte level isprovided and visually displayed. Examples of suitable power sources andmeters may be found, for example, in U.S. Pat. Nos. 4,963,814,4,999,632, and 4,999,582 to Parks et al., U.S. Pat. No. 5,243,516 toWhite et al., and European Patent Application No. 89116797.5, to Hill etal. The disclosures of these patents are incorporated by herein byreference in their entirety.

[0110] A small sample of blood or other aqueous fluid is then applied tothe test device. The current is measured about 10 to about 30 secondsafter applying the sample. The current is read by the meter between theworking and counter electrode and, optionally, is compared to thereference electrode, if it is present. The meter then applies thealgorithm to the current measurement and converts the measurement to ananalyte concentration. This analyte level is visually displayed on themeter.

[0111] From the foregoing description, various modifications and changesin the electrochemical test devices, processes and methods of thisinvention will occur to those skilled in the art. All such modificationscoming within the scope of the appended claims are intended to beincluded therein.

[0112] Additional materials and methods useful in this invention aredisclosed in copending applications Ser. No. 60/019,864 filed Jun. 17,1996 and Ser. No. 08/876,812 filed Jun. 17, 1997, the disclosures ofwhich are incorporated herein by reference in their entirety.

1. An electrochemical test device for determining the presence orconcentration of an analyte in an aqueous fluid sample, saidelectrochemical test device comprising: (a) a non-conductive membranehaving a first skin and a second skin and a matrix therebetween; (b) aworking electrode comprising an amorphous semiconductor material affixedto the non-conductive membrane first skin, said working electrode havingan first electrode area, a first lead and a first contact pad; (c) anopposing electrode comprising an amorphous semiconductor materialaffixed to the non-conductive membrane second skin, said opposingelectrode having an second electrode area, a second lead and a secondcontact pad; and (d) a reagent capable of reacting with the analyte toproduce a measurable change in potential which can be correlated to theconcentration of the analyte in the fluid sample, said reagent imbibedin the membrane matrix between the working electrode and opposingelectrode.
 2. The electrochemical test device of claim 1 wherein saiddevice further comprises a reference electrode comprising an amorphoussemiconductor material affixed to the non-conductive membrane secondskin, said reference electrode having a third electrode area, a thirdlead, and a third contact pad, and wherein at least a portion of thethird electrode area is overlaid with a reference material.
 3. Theelectrochemical test device of claim 2 wherein said reference materialis silver/silver chloride.
 4. The electrochemical test device of claim 1wherein the non conductive membrane material is selected frompolysulphone, polyethersulphone, or nylon and is cased with a tight poreskin on each side and a relatively isotropic matrix between the skinsurfaces.
 5. The electrochemical test device of claim 1 wherein theamorphous semiconductor material is amorphous silicon oxide.
 6. Theelectrochemical test device of claim 1 wherein the reagent comprises aglucose oxidase and a redox mediator.
 7. A method for determining thepresence or concentration of an analyte in an aqueous fluid sample, saidmethod comprising: (a) providing an electrochemical test devicecomprising: (i) a non-conductive double skinned membrane; (ii) a workingelectrode comprising an amorphous semiconductor material affixed to thenon-conductive membranes first skin surface, said working electrodehaving an first electrode area, a first lead and a first contact pad;(iii) a counter electrode comprising an amorphous semiconductor materialaffixed to the non-conductive membranes second skin surface, saidcounter electrode having a second electrode area, a second lead, and asecond contact pad; and (iv) a reagent capable of reacting with theanalyte to produce a measurable change in potential which can becorrelated to the presence or concentration of the analyte in the fluidsample, said reagent imbibed into the membrane between the working andopposing; (b) inserting the electrochemical test device into a meterdevice; (c) applying a sample of an aqueous fluid to the first electrodearea of the working electrode; (d) reading the meter device to determinethe presence or concentration of the analyte in the fluid sample.
 8. Themethod of claim 7 wherein the electrochemical test device furthercomprises a reference electrode comprising an amorphous semiconductormaterial affixed to the non-conductive membranes second surface, saidreference electrode having a third electrode area, a third lead, and athird contact pad, and wherein at least a portion of the third electrodearea is overlaid with a reference material.
 9. The electrochemical testdevice of claim 7 wherein the non conductive membrane material isselected from polysulphone, polyethersulphone, or nylon and is casedwith a tight pore skin on each side and a relatively isotropic matrixbetween each skin surface.
 10. The method of claim 7 wherein the reagentcomprises an enzyme and a redox mediator.
 11. A process for preparing anelectrochemical test device suitable for determining the presence orconcentration of an analyte in an aqueous fluid sample, said processcomprising the steps of: (a) providing a non-conductive double skinnedmembrane; (b) depositing an amorphous semiconductor material on saidfirst skin surface to form a conductive layer which forms a workingelectrode comprising a first electrode having a first electrode area, afirst lead and a first contact pad (c) depositing an amorphoussemiconductor material on said second skin surface to form a conductivelayer, which forms an opposing electrode comprising a second electrodehaving a second electrode area, a second lead and a second contact pad(d) applying a reagent to imbibe the membrane through the fast electrodearea of the working electrode, said reagent being capable of reactingwith an analyte in an aqueous fluid sample to produce a measurablechange in potential which can be correlated to the concentration of theanalyte in the fluid sample.
 12. The process of claim 11 wherein step(c) further comprises forming a reference electrode on the second skinsurface comprising a third electrode having a third electrode area, athird lead and a third contact pad.
 13. The electrochemical test deviceof claim 11 wherein the non conductive membrane material is selectedfrom polysulphone, polyethersulphone, or nylon and is cased with a tightpore skin on each side and a relatively isotropic matrix between eachskin surface.
 14. The process of claim 11 wherein the reagent comprisesa glucose oxidase and a redox mediator.
 15. A process of claim 12wherein said process is continuous.